Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a family of enveloped positive-strand RNA viruses called arteri viruses. Other viruses in this family are the prototype virus, equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV) (de Vries et al., 1997 for review). Striking features common to the Coronaviridae and Arteriviridae have recently resulted in their placement in a newly created order, Nidovirales (Pringle, 1996; Cavanagh, 1997; de Vries et al., 1997). The four members of the Arterivirus group, while being similar in genome organization, replication strategy and amino acid sequence of the proteins are also similar in their preference for infection of macrophages, both in vivo and in vitro (Conzelmann et al., 1993; Meulenberg et al, 1993a).
The genome organization of arteriviruses is reviewed in de Vries et al. (1997). The genome RNA is single-stranded, infectious, polyadenylated and 5′ capped. The genome of PRRSV is small, at 15,088 bases. Both the EAV and LDV genomes are slightly smaller at 12,700 bases and 14,200 bases, respectively. Complete sequences of EAV, LDV and PRRSV genomes are available (Den Boon et al, 1991; Godeny et al, 1993; Meulenberg et al. 1993a).
The genome contains eight open reading frames (ORFs) that encode, in the following order, the replicase genes (ORFs 1a and 1b), the envelope proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF 7) (Meulenberg et al. 1993a). ORFs 2 to 7 are expressed from six sub-genomic RNAs, which are synthesized during replication (Meng et al., 1994, 1996). These sub-genomic RNAs form a 3′ co-terminal nested set and are composed of a common leader, derived from the 5′ end of the viral genome (Meulenberg et al. 1993b). Although the RNAs are structurally polycistronic, translation is restricted to the unique 5′ sequences not present in the next smaller RNA of the set. Two large overlapping open reading frames (ORFs), designated ORF 1a and ORF 1b, take up more than two thirds of the genome. The second ORF, ORF 1b is only expressed after a translational read-through via a −1 frame shift mediated by a pseudoknot structure (Brierley 1995). The polypeptides encoded by these ORFs are proteolytically cleaved by virus-encoded proteases to yield the proteins involved in RNA synthesis.
ORF 2 encodes a 29-30 kDa N-glycosylated structural protein (GP2 or GS) showing the features of a class 1 integral membrane glycoproteins (Meulenberg and Petersen-den Besten, 1996 using the Ter Huurne strain of Lelystad virus). The ORF 2 protein shows 63% amino acid homology when the American VR-2332 isolate is compared to Lelystad virus (Murtaugh et al., 1995). ORF 3 encodes a N-glycosylated 45-50 kDa minor structural protein designated GP3 (van Nieuwstadt et al., 1996). ORF 4 encodes a 31-35 kDA minor N-glycosylated membrane protein designated GP4 (van Nieuwstadt et al., 1996). ORF 5 encodes GP5 or GL, which is a 25 kDA major envelope glycoprotein (Meulenberg et al., 1995). ORF 6 encodes an 18 kDA class III non-glycosylated integral membrane (M) protein (Meulenberg et al, 1995). ORF 7 encodes a 15 kDa non-glycosylated basic protein. Equine arteritis virus (EAV) genome ORF was designated 2a and codes for an essential 8 kDa structural protein called “E” (Snijder et al, 1999). In PRRSV, the homologous ORF has been designated 2b, the ORF 2 coding for GP2 (see above) being renamed ORF 2a (Snijder et al., 1999).
Two main groups of clinical signs are associated with the occurrence of PRRS although it is now recognized that clinical effects vary greatly among infected herds and in many cases, infection is sub-clinical and productivity is within acceptable parameters. The two groups are: (1) Reproductive signs which include premature births, late-term abortions, piglets born weak and increased numbers of still-births and mummifications (Done and Paton, 1995). (2) Signs of respiratory disease are also important in neonatal pigs with labored breathing and coughing being the most dominant characteristics. The symptoms usually occur in pigs about three weeks of age though all ages are susceptible. In contrast to the reproductive failures, clinically overt respiratory disease is harder to reproduce experimentally (Zimmermann et al. 1997). These clinical signs vary considerably and may be influenced by the virus strain (Halbur et al., 1995), age at infection and differences in genetic susceptibility (Halbur et al., 1992), concurrent infections (Galina et al., 1994), pig density, pig movements and housing systems (Done et al., 1996) and immune status including the presence of low levels of PRRS virus-specific antibodies which may be enhancing (Yoon et al., 1994).
There appear to be three routes of transmission: (1) nose to nose or close contact (Done et al, 1996), (2) aerosols (Le Potier et al, 1995), and (3) spread through urine, feces and semen. Transmission via insemination with contaminated semen is well-documented (Yeager et al., 1993; Albina, 1997). In terms of pathogenesis, the most significant change induced by PRRSV is the severe damage to alveolar macrophages, which are destroyed in huge numbers (reviewed in Done and Paton, 1995; Rossow, 1998). The induction of apoptosis in a large number of mononuclear cells in the lungs and lymph nodes might be an explanation for a dramatic reduction in the number of alveolar macrophages and circulating lymphocytes and monocytes in PRRSV-infected pigs (Sirinarumitr et al., 1998; Sur et al., 1998). Coupled with the destruction of circulating lymphocytes and the destruction of the mucociliary clearance system, this may suppress immunity and render pigs more susceptible to secondary infection. An enhanced rate of bacterial secondary infections has been documented following PRRSV infection (Galina et al, 1994; Done and Paton, 1995; Nakamine et al. 1998). The severity of PRRSV infection may be also increased by bacterial or mycoplasma infection (Thacker et al. 1999). In addition a number of viral infections have been found associated with PRRS (Carlson, 1992; Brun et al., 1992; Halbur et al, 1993; Done et al., 1996; Heinen et al., 1998).
Infection with PRRSV usually induces slow and weak anti-viral immune responses, leading to persistent infection and immunosuppression in the lungs of infected pigs. The reported PRRSV immune evasion strategies include inhibition of innate immune responses, induction systemic immunosuppressive cytokine; IL-10 and porcine Tregs (CD4+CD25+Foxp3+ lymphocytes) that resulted in generalized immunosuppression during an early phase of infection. The adaptive immunity against PRRSV is often slow and inefficient, with evidence of polyclonal B cell activation and induction of ADE in the following exposure. Applicants have recently generated experimental evidence suggesting that the immunomodulatory properties of the virus may rely on the interaction of the structural protein and the immune cells (S. Suradhat, unpublished observation). In general, PRRSV infection does not kill the infected pigs, but rather causes several health complications related to suboptimal immune function. Several reports demonstrate that the PRRSV-induced immunomodulatory activities could result in secondary immunodeficiency causing persistent infection, secondary complications, and vaccine failure in the infected pigs.
Although, several commercial vaccines are available in the market, the benefit of vaccine-induced immunity in the vaccinated pigs has not been satisfactory. The modified live vaccine (MLV) has proven more efficacious than the inactivated vaccine due to its ability to induce relatively broader immunity. Evidence also suggest a role for cell-mediated immunity in limiting PRRSV infection and spreading within infected pigs. However, induction of specific immunity by MLV has proven to be delayed and inefficient. In addition, the immunity induced by MLV provides only partial protection against heterologous PRRSV infection. In some cases, the use of MLV has raised concerns regarding safety and induction of immunotolerance.
In general, the development of vaccine against viral infection relies on induction of viral-specific protective humoral and cellular-mediated immunity. The development of effective PRRS vaccine has been extensively challenged with the high antigenic variability of the virus (quasispecies) and its ability to control the immune system via several immunomodulatory activities. Therefore, despite of being properly primed prior to infection, the vaccine-induced, PRRSV-specific effector/memory cells might not be able to function well during an early phase of infection. Since PRRSV alone does not kill infected pigs, we hypothesize that if the PRRSV-induced immunomodulatory effects is removed/reduced, the immune system of the infected host should be able to limit/clear viral infection by itself. This will also help minimizing persistent infection and secondary complications in the late stage of infection.
A vaccine that could induce strong cross-reactive, anti-PRRSV cellular immunity should have benefit on reduction of viremia, PRRSV-induced clinical signs, and improving of the general health condition by reducing secondary complications related to PRRSV-induced immunodeficiency. In addition, avoiding of unnecessary B cell activation by the vaccine antigen would be ideal for implementation of the differentiation of infected and vaccinated animals (DIVA) strategies in the farms. It has been proposed to use needle-free injectors in veterinary field (WO-A-98/03659; WO-A-92/15330; WO-A-98/03658; van Rooij et al., Vet. Immunol. Immunopathol., 1998, 66(2), 113-126; U.S. Pat. No. 6,451,770; Schrijver et al., Vaccine, 1998, 16(2-3), 130-134), but the prior art contains inconsistent and contradictory results (McKercher P. D. et al., Can. J. Comp. Med., 1976, 40, 67-74; Epstein, Hum. Gene Ther., 2002, 13(13), 275-280; Haensler, Vaccine, 1999, 17(7-8), 628-638). Therefore, a skilled person cannot predict whether needle-free delivery will be efficacious for an untested host/vaccine combination.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.